The conduction of electrical signals along myelinated nerve fibers depends on high-density clusters of ion channels at nodes of Ranvier. Demyelinating diseases or injuries disrupt ion channel clusters, block conduction of action potentials in axons, and lead to nervous system dysfunction. Neuron-glia interactions at paranodal junctions flanking nodes contribute to the formation and maintenance of nodes, and participate in bi-directional signaling between neurons and glia. The specific aims of this project are to determine the molecular mechanisms underlying formation and maintenance of 1) nodes of Ranvier and 2) paranodal junctions. A major impediment to these aims has been the relatively few proteins known to be at these sites. We identified a specialized paranodal cytoskeleton and will determine its role in organizing and maintaining neuroglial interactions and axon stability. We will ablate components of the paranodal cytoskeleton using in vivo electroporation to deliver shRNA plasmids. We speculate that a major cause of axon degeneration as seen in demyelinating diseases is disruption of the axonal cytoskeleton and altered neuroglial interactions. We will identify additional paranodal proteins using biochemical and proteomic methods. To determine the mechanisms underlying node of Ranvier formation, we will identify critical proteins, protein domains, protein-protein interactions, and nodal localization determinants. The proposed experiments are important for many nervous system diseases where neuron-glia interactions are disrupted (e.g., multiple sclerosis, Guillain-Barre syndrome, spinal cord injury). Identification of the mechanisms underlying neuron-glia interactions may contribute to better therapeutic treatments designed to preserve these interactions and their contribution to neuronal health and function. PERFORMANCE SITE(S) (organization, city, state) University of Connecticut Health Center Farmington, CT PHS 398 (Rev. 04/06) Page 2 Form Page 2 Principal Investigator/Program Director (Last, First, Middle): Rasband, Matthew, Neil KEY PERSONNEL. See instructions. Use continuation pages as needed to provide the required information in the format shown below. Start with Principal Investigators). List all other key personnel in alphabetical order, last name first. Name eRA Commons User Name Organization Role on Project Rasband, Matthew mrasband UCHC Principal Investigator Hedstrom, Kristian UCHC Graduate Student Ogawa, Yasuhiro UCHC Postdoctoral Fellow Schafer, Dorothy UCHC Graduate Student Susuki, Keiichiro UCHC Postdoctoral Fellow OTHER SIGNIFICANT CONTRIBUTORS Name Organization Role on Project Human Embryonic Stem Cells 3 No Q Yes If the proposed project Involves human embryonic stem cells, list below the registration number of the specific cell llne(s) from the following list: http://stemcells.nih.gov/registrv/index.asp. Use continuation pages as needed. If a specific line cannot be referenced at this time, include a statement that one from the Registry will be used. Cell Line PHS 398 (Rev. 04/06) Page 3 Form Page 2-continued Number the following pages consecutively throughout the aoolication. Do not use suffixes such as 4a. 4b. Principal Investigator/Program Director (Last, First, Middle): Rasband, Matthew, Neil The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page. RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page 1 Description, Performance Sites, Key Personnel, Other Significant Contributors, and Human Embryonic Stem Cells 2-3 Table of Contents Detailed Budget for Initial Budget Period (or Modular Budget) Budget for Entire Proposed Period of Support (notapplicable with Modular Budget) Budgets Pertaining to Consortium/Contractual Arrangements (notapplicable with Modular Budget) Biographical Sketch - Principal Investigator/Program Director (Not to exceed four pages) 6-8 Other Biographical Sketches (Notto exceed four pages for each - Seeinstructions) 9-14 Resources 15 Research Plan. 16-43 Introduction to Revised/Resubmission Application (Notto exceed 3 pages.)... 16-18 Introduction to Supplemental/Revision Application (Notto exceed one page.).. A. Specific Aims B. Background and Significance 20-22 C. Preliminary Studies/Progress Report (Items A-D:not to exceed 25 pages). 22-35 D. Research Design and Methods 35-43 E. Human Subjects Research 44 Protection of Human Subjects (Required if Item 4 on the Face Page is marked Yes) Data and Safety Monitoring Plan (Required if Item 4 on the Face Page is marked 'Yesand a Phase I, II, or III clinical trial is proposed) Inclusion of Women and Minorities (Required if Item 4 on the Face Page is marked Yes and is Clinical Research) Targeted/Planned Enrollment Table (for new and continuing clinical research studies) Inclusion of Children (Required if Item 4 on the Face Page is marked Yes) F. Vertebrate Animals 44 G. Select Agent Research 44 H. Literature Cited 45-50 I. Multiple PI Leadership Plan 50 J. Consortium/Contractual Arrangements 50 K. Resource Sharing 50 L Letters of Support (e.g., Consultants) Checklist. Appendix (Five collated sets. Nopage numbering necessary for Appendix.) Check if Appendix is Included Number of publications and manuscripts accepted for publication (not to exceed 10) 10 Other items (list): PHS 398 (Rev. 04/06) Paae4 Form Pane 3